The resurgence of mosquito-borne diseases is having a devastating impact on global health and an urgent need exists to develop new control strategies. A new strategy being actively investigated is the use of transgenics for the control of vector-borne diseases, i.e., to change vector competence by genetic transformation to reduce the ability of mosquitoes within a population to transmit a particular pathogen. However, if we are to evaluate the use of mosquito transformation as a viable disease control methodology it is imperative that we gain a better understanding of the mosquito's innate immune response that governs vector competence. Melanotic encapsulation is a cellular innate immune response that is responsible for the resistant phenotype of mosquitoes to filarial worms and malaria parasites. Through support from our parent grant (Immune Response of Mosquitoes to Filarial Worms) we are studying the biochemistry and genetic regulation of melanotic encapsulation, but during the course of these studies we have determined that detailed studies of gene expression in the circulating immune reactive cells (hemocytes) of mosquitoes is necessary if we are to clarify mechanisms of immune recognition and the initiation of defense responses. Very few hemocytes (<1,200) are present in an individual mosquito and they are not amenable to in vitro culture or even short-term in vitro maintenance; consequently, we have constructed cDNA libraries representing mRNA obtained from immune-activated hemocytes from two mosquito species. Generation of a limited number of ESTs from these libraries indicated a low percentage of ribosomal clones and revealed a number of matches to proteins with known innate immunity functions. These data convinced us that the systematic development of additional ESTs would facilitate the construction of informative microarrays that could be used to test specific hypotheses about the role hemocytes play in influencing vector competence. The budget of the parent grant was not designed to cover the costs required to generate the number of ESTs required, let alone the costs associated with oligonucleotide microarray construction; and, importantly, we could not have anticipated the feasibility of a comprehensive transcriptome approach to assess gene regulation in our model. The specific aims of the parent grant would be significantly enhanced by the multi-dimensional data from these additional tools. It is therefore appropriate to use the R21 mechanism for technology application to enhance our NIAID-funded research by (1) generating a large EST data set from immune-activated hemocyte cDNA libraries, (2) constructing oligonucleotide microarrays representing these ESTs, and (3) using these microarrays in data mining of hemocyte expression profiles to identify distinct patterns of transcription underlying the immune response of mosquitoes against filarial worms.